The broad, long-range objective of this project is the rational design of contraceptive strategies based on the reversible arrest of spermatogonia (Spg) in the vitamin A-deficient (VAD) testis. Our intent is to uncover specific mechanisms that reversibly arrest Spg in this model. Of the two pertinent cell types (arrested Spg and Sertoli cells) in the epithelium of the VAD testis, only the Sertoli cell expresses significant amounts of retinoic acid receptor-alpha (RARalpha). Thus, our overall hypothesis: paracrine signal(s) from Sertoli cells mediate the effects of vitamin A on Spg. Our working hypothesis is that Sertoli cell-derived activin arrests Spg in the VAD testis and that vitamin A rescues proliferation by down-regulating activin and up-regulating its binding protein follistatin-like protein. The objectives of this initial funding period are to identify future contraceptive targets by determining the role specific signaling molecules and pathways play during the vitamin A-dependent regulation of Spg proliferation. We intend to accomplish this by completing the following specific aims: (1) Test our prediction that vitamin A regulates specific signaling factors (activin, inhibin), binding proteins (follistatin, follistatin-related protein) and receptors (activin receptors i & ii). (2) Determine whether the vitamin a status of the testis regulates smad-dependent signaling pathways downstream of the activin receptor and synthesis of cyclin-dependent kinase inhibitor(s) to effect changes in the spermatogonial cell cycle. (3) Test the efficacy of candidate factors to regulate the proliferation of Spg and test our hypothesis that activin-dependent signaling regulates cyclin-dependent kinase inhibitors to effect these changes. (4) Identify additional paracrine factors and receptors that participate in the reversible VAD arrest of spermatogonia. Completing these aims will define specific mechanisms that reverse an intrinsic checkpoint in the genetic program directing spermatogonial proliferation, a significant advance towards our goal of designing a successful male contraceptive.